It is our objective to carry out detailed structure analyses of enzymes by the established techniques of x-ray crystallography. We propose to continue studies of the flavoprotein enzymes involved in biological oxidations, already initiated by the determination of the crystal structures of two oxidation states of clostridial flavodoxin. Our choice of enzymes appropriate for structure analysis has been determined by the desire to 1) elucidate enzyme-substrate interactions, 2) understand the changes of structure accompanying oxidation or reduction of the prosthetic group, 3) correlate the structure with biological reactivity and physical properties. The results already obtained with flavodoxin, a prototype flavoprotein dehydrogenase, have led to a description of the flavin- protein interactions, shown subtle changes of structure upon reduction of the flavin, and revealed unexpected species variations in the active center (1) along with conservation of an elemental dehydrogenase structure (2). Completion of the structural analysis of flavodoxin is proposed. As a major goal, we would like to extend structural analyses to larger flavo-proteins such as those for which the flavodoxins (or similar electron carriers) are substrates, viz. ferredoxin-NADP- reductase or adrenodoxin reductase. We will attempt to crystallize complexes containing both reductase and electron carrier. An alternative subject for study of E-S interaction is lipoamide hydrogenase. Although crystallization of some of these proteins has been reported, a major effort to obtain suitable crystals will be required. In parallel with the structure work we intend to study the spectral properties of the above proteins, both in solution and crystalline states. It will be our aim to compare the properties of the crystalline E-S "models" with deductions from kinetic and spectroscopic studies in solution. We have also undertaken the crystal structure analyses of small molecules related to the proteins under investigation.